The membrane attack complex of complement C5b-9 is responsible for complement mediated tissue damage. The formed complex can be specifically identified by neoantigens which are not present on isolated components within the complex. We had previously prepared goat polyclonal antiserum to C5b-9 neoantigens. The antiserum was absorbed extensively against pooled normal human serum which had been coupled to a solid phase matrix. The absorption procedure removes antibodies reacting with native individual constituents (C5, C6, C7, C8 and C9) making up the C5b-9 complex, but does not remove antibodies to neo-antigenic determinants. To characterize the antiserum, an ELISA was set up to detect C5b-9 neoantigens. Serum containing I-125C9 was treated with zymosan to activate complement, then subjected to sucrose density gradient ultracentrifugation. Gradient fractions were used to coat ELISA plates, and detection of neoantigen in zymosan-activated but not control serum was accomplished. Anti-neoantigen antisera were also used in immunofluorescence studies to localize C5b-9 on complement-lysed erythrocytes and tissues from patients with systemic lupus erythematosis. Attempts to prepare a monoclonal antibody to C5b-9 neoantigens have not yet been successful. In the second phase of the project, hydrophilic and hydrophobic fragments of C9 were produced by -thrombin cleavage, then separated and purified by chromatography on hydroxylapatite in 0.1% SDS. Three monoclonals raised against C9 were shown to react specifically with the hydrophobic but not the hydrophilic fragment, as assessed by Western blot. Work in progress is directed toward generating a monoclonal to the hydrophilic fragment of C9.